title: A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties.
abstract: Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
authors.affiliation: ‡Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Qingwei
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: *The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.
authors.forename: Samuel N
authors.initials: SN
authors.lastname: Young
authors.affiliation: §Department of Microbiology and Immunology, Stanford University, Stanford, CA 24305-5124, U.S.A.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Reese
authors.affiliation: ¶Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, U.K.
authors.forename: Fiona P
authors.initials: FP
authors.lastname: Bailey
authors.affiliation: ¶Department of Biochemistry, Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, U.K.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Eyers
authors.affiliation: ∥School of Medicine and Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere 33014, Finland.
authors.forename: Daniela
authors.initials: D
authors.lastname: Ungureanu
authors.affiliation: ∥School of Medicine and Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere 33014, Finland.
authors.forename: Henrik
authors.initials: H
authors.lastname: Hammaren
authors.affiliation: ∥School of Medicine and Institute of Biomedical Technology, University of Tampere and Tampere University Hospital, Tampere 33014, Finland.
authors.forename: Olli
authors.initials: O
authors.lastname: Silvennoinen
authors.forename: Leila N
authors.initials: LN
authors.lastname: Varghese
authors.forename: Kelan
authors.initials: K
authors.lastname: Chen
authors.affiliation: *The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia.
authors.forename: Anne
authors.initials: A
authors.lastname: Tripaydonis
authors.affiliation: **Cardiovascular Research Institute and Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158-9001, U.S.A.
authors.forename: Natalia
authors.initials: N
authors.lastname: Jura
authors.affiliation: ††Department of Molecular Cardiology, Lerner Research Institute, NB20, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A.
authors.forename: Koichi
authors.initials: K
authors.lastname: Fukuda
authors.affiliation: ††Department of Molecular Cardiology, Lerner Research Institute, NB20, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A.
authors.forename: Jun
authors.initials: J
authors.lastname: Qin
authors.affiliation: ‡‡Department of Biology, California Institute of Technology, Pasadena, CA 91125, U.S.A.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Nimchuk
authors.affiliation: §§Department of Biology, Stanford University, Stanford, CA 24305-5020, U.S.A.
authors.forename: Mary Beth
authors.initials: MB
authors.lastname: Mudgett
authors.affiliation: ¶¶Centre de Recherche du Centre Hospitalier Universitaire de Québec and and Faculté de Médicine, Département de Pédiatrie, Université Laval, Québec G1V 4G2, Canada.
authors.forename: Sabine
authors.initials: S
authors.lastname: Elowe
authors.affiliation: ∥∥Australian Synchrotron, Clayton, Victoria 3168, Australia.
authors.forename: Christine L
authors.initials: CL
authors.lastname: Gee
authors.affiliation: ***Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: Ling
authors.initials: L
authors.lastname: Liu
authors.affiliation: ***Cancer Research Program, The Kinghorn Cancer Centre, Garvan Institute of Medical Research, 370 Victoria Street, Darlinghurst, Sydney, NSW 2010, Australia.
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Daly
authors.affiliation: †††Genentech, 1 DNA Way, MS 93, South San Francisco, CA 94010, U.S.A.
authors.forename: Gerard
authors.initials: G
authors.lastname: Manning
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Babon
authors.affiliation: ‡Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Isabelle S
authors.initials: IS
authors.lastname: Lucet
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D007313:Insecta; D053614:Janus Kinase 2 / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020893:Receptor, ErbB-3 / Q000737:chemistry* / Q000145:classification* / Q000235:genetics
chemical_list: C581292:ERBB3 protein, human; D020893:Receptor, ErbB-3; C507924:JAK2 protein, human; D053614:Janus Kinase 2